Killing Two Birds with One Stone: Discovery of Dual Inhibitors of Oxygen and Fumarate Respiration in Zoonotic Parasite, Echinococcus multilocularis.

Ascofuranone (AF), a meroterpenoid isolated from various filamentous fungi, including Acremonium egyptiacum, has been reported as a potential lead candidate for drug development against parasites and cancer. In this study, we demonstrated that AF and its derivatives are potent anthelminthic agents, particularly against Echinococcus multilocularis, which is the causative agent of alveolar echinococcosis. We measured the inhibitory activities of AF and its derivatives on the mitochondrial aerobic and anaerobic respiratory systems of E. multilocularis larvae. Several derivatives inhibited complex II (succinate:quinone reductase [SQR]; IC50 = 0.037 to 0.135 µM) and also complex I to III (NADH:cytochrome c reductase; IC50 = 0.008 to 0.401 µM), but not complex I (NADH:quinone reductase), indicating that mitochondrial complexes II and III are the targets. In particular, complex II inhibition in the anaerobic pathway was notable because E. multilocularis employs NADH:fumarate reductase (fumarate respiration), in addition to NADH oxidase (oxygen respiration), resulting in complete shutdown of ATP synthesis by oxidative phosphorylation. A structure-activity relationship study of E. multilocularis complex II revealed that the functional groups of AF are essential for inhibition. Binding mode prediction of AF derivatives to complex II indicated potential hydrophobic and hydrogen bond interactions between AF derivatives and amino acid residues within the quinone binding site. Ex vivo culture assays revealed that AF derivatives progressively reduced the viability of protoscoleces under both aerobic and anaerobic conditions. These findings confirm that AF and its derivatives are the first dual inhibitors of fumarate and oxygen respiration in E. multilocularis and are potential lead compounds in the development of anti-echinococcal drugs.

Anthelmintic Baiting of Foxes against Echinococcus multilocularis in Small Public Area, Japan.

We distributed anthelmintic baits on a university campus in Japan inhabited by foxes infected with Echinococcus multilocularis to design an effective baiting protocol for small public areas. High-density baiting can reduce the risk for human exposure to the parasite to near zero. However, monthly baiting is recommended to maintain this effect.

In vivo efficacy of combination therapy with albendazole and atovaquone against primary hydatid cysts in mice.

Alveolar echinococcosis (AE) is caused by the larval stage of Echinococcus multilocularis. Chemotherapy for AE involves albendazole (ABZ), which has shown insufficient efficacy. More effective chemotherapy for AE is needed. Previously, we have demonstrated that atovaquone (ATV), an antimalarial, inhibits mitochondrial complex III of E. multilocularis and restricts the development of larval cysts in in vivo experiments. Therefore, in this study, we evaluated the efficacy of ABZ and ATV combination therapy on E. multilocularis in culture and in vivo experiments. Protoscoleces were treated with 50 µM ABZ and/or ATV in the medium; the duration of parasite elimination was determined under aerobic and anaerobic culture. In the in vivo experiment, the effects of ABZ and ATV combination treatment in BALB/c mice infected orally with eggs from the feces of an adult-stage E. multilocularis-infected dog were compared with those of standard oral ABZ therapy. In the culture assay, the duration of elimination associated with ABZ and ATV combination treatment was shorter than that associated with ATV alone under aerobic conditions. Protoscolex viability progressively reduced owing to the combination treatment under anaerobic conditions; however, either drug used singly did not exhibit antiparasitic effects under hypoxia. Furthermore, compared with ABZ alone, the combination treatment significantly reduced the growth of the primary cyst in the liver of mice infected orally with parasite eggs (P = .011). ATV enhances the effect of ABZ in the treatment of AE in mice.

Diagnosis of canine Echinococcus multilocularis infections by copro-DNA tests: comparison of DNA extraction techniques and evaluation of diagnostic deworming.

The use of copro-DNA detection methods for the diagnosis of canine Echinococcus multilocularis infection was evaluated with a focus on DNA extraction techniques: two commercial kits and a modified alkaline-sodium dodecyl sulfate (SDS) technique. Dog feces (0.2 g) mixed with a protoscolex or with 1 or 10 eggs of E. multilocularis were subjected to DNA detection following extraction by these methods. DNA was extracted from all protoscolex samples by all methods, but success for samples with eggs depended on extraction technique with the modified technique showing success on all samples. Following experimental infection of dogs, copro-DNA was successfully extracted from fecal samples (0.2 g) of dogs in the patent period by all methods. In the prepatent period, PCR testing of feces subsamples (0.2 g) extracted by each technique was positive at a rate of 79.6-94.4%. Extraction by the modified technique with fecal samples of over 1 g showed detection of copro-DNA in all samples in both the patent and prepatent periods, and it produced reproducible detection in the addition recovery test using feces from 72 different domestic dogs. As copro-DNA was detected for at least 1 day following deworming with administration of anthelmintic drugs in experimentally infected dogs, diagnostic deworming might be useful for clinical examination. Using the present detection method can provide quick and accurate diagnosis of canine E. multilocularis infection, which with prompt management and treatment of infected dogs can prevent pet owners from becoming infected and prevent echinococcosis from spreading into non-endemic areas.

Mitogenomic exploration supports the historical hypothesis of anthropogenic diffusion of a zoonotic parasite Echinococcus multilocularis.

Animal movement across regions owing to human activity can lead to the introduction of pathogens, resulting in disease epidemics with medical and socioeconomic significance. Here, we validated the hypothesis that human activity, such as the transportation of infected animals, has played a significant role in introducing the zoonotic parasite Echinococcus multilocularis into Hokkaido, Japan, by synthesizing and evaluating parasite genetic data in light of historical records. Our analysis indicates that a major genetic group in Hokkaido originated from St. Lawrence Island, USA, which is in accordance with the route suggested by historical descriptions. Moreover, we identified a minor genetic group closely related to parasites found in Sichuan, China. This fact implies that parasite invasion in Japan may result from complex and inadvertent animal translocations. These findings emphasize the anthropogenic impacts on zoonotic parasite spread and provide a crucial perspective for preventing future potential epidemics.

Sensitivity comparison between Mini-FLOTAC and conventional techniques for the detection of Echinococcus multilocularis eggs.

Canines serve as the definitive host of Echinococcus multilocularis. This study evaluated the sensitivity of the Mini-FLOTAC technique (MF) for the detection of E. multilocularis eggs in definitive hosts. First, we investigated the effects of heat inactivation and preservative conditions on the detection rate of eggs obtained from experimentally infected dogs. The sensitivity of MF was compared with that of eight other techniques: the centrifugal flotation with sucrose or zinc sulfate, MGL, AMS III, and a combination of MF and flotation/sedimentation techniques. Finally, we compared the sensitivity of MF and the centrifugal flotation with sucrose for the feces of E. multilocularis-infected foxes. The detection rate reached a plateau level with a specific gravity (s.g.) 1.22 for fresh eggs, but the highest rates were obtained with s.g. greater than 1.32 for heat-inactivated eggs. There was no significant difference in the detection rate among the preservative conditions. MF showed significantly higher EPG than the other techniques. Moreover, it showed higher diagnostic sensitivity for the fox feces than the centrifugal flotation technique. These results suggest that heat inactivation may alter s.g. of E. multilocularis eggs and that MF with zinc sulfate (s.g. = 1.32) would be effective for detecting heat-inactivated E. multilocularis eggs.

Data on the combined effect of atovaquone, mefloquine, and 3-bromopyruvic acid against Echinococcus multilocularis protoscoleces.

The dataset presented here is related to a previous research article titled "Mitochondrial Complex III in Larval Stage of Echinococcus multilocularis as a Potential Chemotherapeutic Target and in vivo Efficacy of Atovaquone Against Primary Hydatid Cysts"[1]. In this report, data were collected from aerobic and anaerobic culture assays of E. multilocularis protoscoleces in the presence of three anti-echinococcal drug candidates (atovaquone, mefloquine, and 3-bromopyruvic acid). The data were analyzed for viability of the protoscoleces between day 0 and day 7 upon adding drug candidates. In aerobic condition, all drug candidates caused damage to the protoscoleces, as described previously [1], [2], [3], [4], [5], [6]. Mefloquine, alone as well as in combination with atovaquone, immediately eliminated the protoscoleces, whereas combination of atovaquone with 3-bromopyruvic acid did not show clear synergy. In anaerobic condition, mefloquine, alone as well as in combination with atovaquone, eliminated protoscoleces immediately. 3-Bromopyruvic acid showed stronger efficacy in anaerobic condition than in aerobic condition. Combination of atovaquone with 3-bromopyruvic acid eliminated the protoscoleces, indicating that synergy occurred only under anaerobic condition. The data clarified that combined use of the three drugs eliminated protoscoleces in both aerobic and anaerobic conditions, hence suggesting that these could inhibit aerobic and anaerobic respiration pathways of Echinococcus multilocularis in vivo. The obtained data would be useful for the development of new drug dosing method for alveolar echinococcosis.

Echinococcus multilocularis: purification and characterization of glycoprotein antigens with serodiagnostic potential for canine infection.

We show that a conventionally purified glycoprotein component of Echinococcus multilocularis protoscolex, designated as Emgp-89, may be useful as a serodiagnostic antigen for detecting E. multilocularis infection in dogs domesticated in endemic areas. Emgp-89 was obtained from the parasite material by a simple procedure using Con A-agarose and subsequent gel filtration chromatography. The purified fraction showed a molecular weight of >4000kDa upon gel filtration and reacted with a series of lectins that specifically bind to mannose, galactose, N-acetylglucosamine, and N-acetylgalactosamine. Subsequently, serodiagnostic performance of Emgp-89 was evaluated through enzyme-linked immunosorbent assays (ELISAs) by using sera from normal, domestic dogs and dogs infected with other helminths. Emgp-89 positively reacted with all 16 serum samples from E. multilocularis-infected dogs, thus showing that this antigen is highly sensitive. On the other hand, the specificity of Emgp-89-based ELISA, determined using 41 serum samples from dogs infected with other helminths, was relatively low (83%). As an attempt to improve the specificity of Emgp-89-based ELISA, we pretreated Emgp-89 with proteinase K or sodium periodate, expecting that these treatments would enable discrimination of true positives from false positives. The ELISA value increased after treatment with sodium periodate in most false-positive samples, whereas significant decreases were observed in sera from all dogs infected with E. multilocularis. Further evaluation of this antigen should be performed using sera from dogs infected with closely-related parasites, including taeniid cestodes, which are expected to prove that this serodiagnostic system is sufficiently specific for clinical and field applications.

Cystic echinococcosis in humans and animals in Egypt: An epidemiological overview.

Cystic echinococcosis (CE), caused by the cestode Echinococcus granulosus (sensu lato), is a serious neglected zoonotic disease in many parts of the world, including Egypt. Thus far, the actual incidence of CE in the Egyptian population remains unknown. Infection with E. granulosus (s.l.) is common among stray dogs in rural and suburban areas owing to the spread of parasite eggs. Herein, we present an updated review of published data on the incidence of CE in humans and animals as well as the genotypes prevalent in Egypt. CE occurs in most parts of Egypt; however, available data are mostly from northern Egypt, particularly Cairo and Giza. In southern Egypt, the disease is likely to be underdiagnosed or underreported. A few risk factors were studied. In the Egyptian population, residency in rural areas, farming, and age were significant factors for acquiring CE. In livestock, age, sex and season have been associated with high prevalence of CE. Several genotypes have been identified among livestock (G1, G4, G5, G6 and G7) and humans (G1, G6 and G7). This literature review underscores the need for a precise national surveillance system to track CE distribution in humans and animals and design appropriate preventive and control strategies for this disease.

Effect of the anti-parasitic compounds pyrvinium pamoate and artemisinin in enzymatic and culture assays: Data on the search for new anti-echinococcal drugs.

The dataset presented herein is related to a previous research article titled "Mitochondrial Complex III in Larval Stage of Echinococcus multilocularis as a Potential Chemotherapeutic Target and in vivo Efficacy of Atovaquone Against Primary Hydatid Cysts" [1]. In this report, data were collected by screening drugs for echinococcosis. We investigated the inhibitory activities of artemisinin and pyrvinium pamoate against the mitochondrial respiratory enzymes in E. multilocularis protoscoleces. Artemisinin did not inhibit mitochondrial complexes I, II, and III. However, pyrvinium pamoate inhibited complex I at 11 µM, although complexes II and III were not inhibited. In the culture assay, E. multilocularis protoscoleces were treated with atovaquone (ATV), rotenone, praziquantel, artemisinin, and pyrvinium pamoate at a final concentration of 50 µM in different culture media. The viability of protoscoleces was compared under aerobic and anaerobic conditions via culture experiments. The survival days of E. multilocularis protoscoleces were evaluated in the drug-treated group compared with those in the non-treated group. The results of these culture assays revealed that praziquantel and artemisinin did not eliminate the protoscoleces under both aerobic and anaerobic conditions. However, a stronger elimination ability was observed with the co-administration of praziquantel or artemisinin with ATV than with ATV alone under aerobic conditions. Pyrvinium pamoate completely killed protoscoleces at 5 and 7 days under aerobic and anaerobic conditions, respectively. Pyrvinium pamoate behaved identically to rotenone, the complex I inhibitor, in the culture treatment assay. The data serve as a reference for the development of novel anti-echinococcal drugs.

Early-phase migration dynamics of Echinococcus multilocularis in two mouse strains showing different infection susceptibilities.

The early-phase migration dynamics of Echinococcus multilocularis in the intermediate hosts remain largely unknown. We compared the parasite burden in the intestine, liver and faeces of DBA/2 and C57BL/6 mouse strains using parasite-specific quantitative PCR. Our results indicated that the parasites invaded mainly from the middle segments of the small intestine and completed migration to the liver within 24 h p.i. C57BL/6 mice had lower parasite DNA burdens in the intestine and liver but higher in the faeces than DBA/2 mice, suggesting that parasite invasion of the intestine may be a critical stage regulating susceptibility to E. multilocularis infection in mice.

Characterization of microRNAs expressed in the cystic legion of the liver of Mus musculus perorally infected with Echinococcus multilocularis Nemuro strain.

Alveolar echinococcosis (AE) is a zoonosis caused by the metacestode of Echinococcus multilocularis. The published genome of E. multilocularis showed that approximately 86% of its genome is non-coding. Micro RNAs (miRNAs) are small non-coding regulatory RNAs, and recent studies on parasitic helminths expect miRNAs as a promising target for drug development and diagnostic markers. Prior to this study, only a few studies reported the E. multilocularis miRNA profiles in the intermediate host. The primary objective of this study was to characterize miRNA profiles via small RNA-seq in E. multilocularis Nemuro strain, a laboratory strain of Asian genotype, using mice perorally infected with the parasite eggs. The data were then compared with two previously published small RNA-seq data. We identified 44 mature miRNAs as E. multilocularis origin out of the 68 mature miRNA sequences registered in the miRNA database miRbase. The highest quantities of miRNAs detected were miR-10-5p, followed by bantam-3p, let-7-5p, miR-61-3p, and miR-71-5p. The top two most abundant miRNAs (miR-10-5p and bantam-3p) accounted for approximately 80.9% of the total parasite miRNAs. The highly expressed miRNA repertoire is mostly comparable to that obtained from the previous experiment using secondary echinococcosis created by an intraperitoneal administration of metacestodes. A detailed characterization and functional annotations of these shared miRNAs will lead to a better understanding of parasitic dynamics, which could provide a basis for the development of novel diagnostic and treatment methods for AE.

Echinococcus multilocularis: two-dimensional Western blotting method for the identification and expression analysis of immunogenic proteins in infected dogs.

Domesticated dogs are an important potential source of Echinococcus multilocularis infection in humans; therefore, new molecular approaches for the prevention of the parasite infection in dogs need to be developed. Here, we identified and characterized an immunogenic protein of the parasite by using a proteome-based approach. The total protein extracted from protoscoleces was subjected to two-dimensional Western blotting with sera from dogs experimentally infected with E. multilocularis. Two protein spots showed major reactivity to the sera from infected dogs. The N-terminal amino acid sequences of these spots were identical to the deduced amino acid sequence of the product of the putative hsp20 gene. RT-PCR and Western blot analyses revealed that the putative hsp20 gene and its products were expressed in almost all stages of the parasite life cycle. Furthermore, recombinant hsp20 showed specific reactivity to the sera from infected dogs, suggesting that this molecule may facilitate the development of a practical vaccine.

Mitochondrial complex III in larval stage of Echinococcus multilocularis as a potential chemotherapeutic target and in vivo efficacy of atovaquone against primary hydatid cysts.

Echinococcus multilocularis employs aerobic and anaerobic respiration pathways for its survival in the specialized environment of the host. Under anaerobic conditions, fumarate respiration has been identified as a promising target for drug development against E. multilocularis larvae, although the relevance of oxidative phosphorylation in its survival remains unclear. Here, we focused on the inhibition of mitochondrial cytochrome bc1 complex (complex III) and evaluated aerobic respiratory activity using mitochondrial fractions from E. multilocularis protoscoleces. An enzymatic assay revealed that the mitochondrial fractions possessed NADH-cytochrome c reductase (mitochondrial complexes I and III) and succinate-cytochrome c reductase (mitochondrial complexes II and III) activities in the aerobic pathway. Enzymatic analysis showed that atovaquone, a commercially available anti-malarial drug, inhibited mitochondrial complex III at 1.5 nM (IC50). In addition, culture experiments revealed the ability of atovaquone to kill protoscoleces under aerobic conditions, but not under anaerobic conditions, indicating that protoscoleces altered their respiration system to oxidative phosphorylation or fumarate respiration depending on the oxygen supply. Furthermore, combined administration of atovaquone with atpenin A5, a quinone binding site inhibitor of complex II, completely killed protoscoleces in the culture. Thus, inhibition of both complex II and complex III was essential for strong antiparasitic effect on E. multilocularis. Additionally, we demonstrated that oral administration of atovaquone significantly reduced primary alveolar hydatid cyst development in the mouse liver, compared with the untreated control, indicating that complex III is a promising target for development of anti-echinococcal drug.

First report of Sarcocystis pilosa sporocysts in feces from red fox, Vulpes vulpes schrencki, in Hokkaido, Japan.

Sarcocysts of various Sarcocystis spp. are highly prevalent in wild sika deer, Cervus nippon yesoensis, in Hokkaido, Japan, and four species have been identified based on morphological and molecular characteristics: S. ovalis, S. pilosa, S. tarandi-like, and S. truncata-like. The definitive hosts of S. ovalis are corvids, but the hosts of the other species have not yet been identified. Aiming to determine the definitive hosts of these species, we collected 65 red fox (Vulpes vulpes schrencki) fecal samples in eastern Hokkaido and examined them for fecal sporocysts using a modified sucrose flotation method. One fecal sample contained typical Sarcocystis sporocysts, which were identified as S. pilosa based on 18S ribosomal RNA and cytochrome c oxidase subunit I gene sequences. This is the first identification of S. pilosa sporocysts in the wild. These findings indicate that red foxes serve as a definitive host of S. pilosa, and that red foxes constitute a source of S. pilosa infection for deer in Hokkaido.

Adult worm exclusion and histological data of dogs repeatedly infected with the cestode Echinococcus multilocularis.

The data presented in this article are related to a previously published research article titled "The timing of worm exclusion in dogs repeatedly infected with the cestode Echinococcus multilocularis" (2016) [1]. This data describe a comparison of worm exclusion in the early stage of infection (1 day and 6 days post-infection) between dogs infected for the first time (control group) and dogs repeatedly infected with the parasite 4 times (repeated infection groups). We observed that 6 days post reinfection, the number of adult worms in repeated-infection groups decreased by 88.7% compared with the control group. Histological analysis comparison of the small intestinal mucosa from healthy, first infected, and repeatedly infected dogs are also reported. We observed no clear pathological abnormality, except the shortening of microvillus in reinfected dogs. However, eosinophil accumulation and eosinophilic ulcers were observed in some reinfected dogs. This data could be useful as preliminary data to develop a final host vaccine for this parasite.

Characterization of emY162 encoding an immunogenic protein cloned from an adult worm-specific cDNA library of Echinococcus multilocularis.

A cDNA library based on mRNA from adult worms of Echinococcus multilocularis was constructed. One cDNA clone, emY162, was isolated from this cDNA library. The putative protein from emY162 cDNA consists of 153 amino acids and has a predicted molecular weight of 17.0 kDa. The amino acid sequences of EMY162 are predicted to be a hydrophobic N-terminus conserving a secretory signal, and a hydrophobic C-terminus encoding a transmembrane domain or glycosyl-phosphatylinositol membrane anchor, and to have single fibronectin type III-like domain. In addition, it was shown that the emY162 gene (1738 bp) in the E. multilocularis genome DNA consists of three exons and two introns, and that emY162 is expressed in all four stages (protoscoleces, cultured metacestodes, immature adult worms and mature adult worms). Moreover, immunity to recombinant EMY162, which comprises the fibronectin type III-like domain on the EMY162 protein, was examined. Immune responses to the recombinant EMY162 were studied by using serum from dogs infected with E. multilocularis. Strong IgG immune responses were detected in Western blots.

High probability of pet dogs encountering the sylvatic cycle of Echinococcus multilocularis in a rural area in Hokkaido, Japan.

Surveillance of Echinococcus multilocularis in 98 pet dogs kept in a rural area of Hokkaido, Japan, from March 2018 to March 2019 suggested infection in seven dogs (7.1%) by E. multilocularis-specific copro-DNA examination, and one of them excreted E. multilocularis eggs that were identified by sequence analyses. Among the infected dogs, three were not allowed to run free when outdoors. Based on detection of E. multilocularis eggs in fox feces collected from roadsides in the same area, dogs kept in rural areas may have a high probability of becoming infected after preying on infected voles along such roadsides, even in domesticated settings. Therefore, examination along with periodic deworming administration is considered necessary to prevent transmission from dogs to owners.

Molecular characterization of three Sarcocystis spp. from wild sika deer (Cervus nippon yesoensis) in Hokkaido, Japan.

Diaphragm samples from 65 hunted sika deer (Cervus nippon yesoensis) from Hokkaido, Japan were examined for the presence of sarcocysts based on histological sections. Morphologically, the detected sarcocysts grouped into three types: (Type 1) 108.0-305.0 µm in width, thick-walled (4.3-7.0 µm) with tombstone-like protrusions; (Type 2) 25.0-69.5 µm in width, thick-walled (3.8-8.0 µm) with finger-like protrusions; and (Type 3) 22.5-55.0 µm in width, thin-walled (under 1 µm) with no visible protrusions under light microscopy. All samples contained at least one sarcocyst type, and multiparasitism was apparent in 58 samples. Morphologically, Type 1 sarcocysts were found in 19 (29.2%) samples, Type 2 in 62 (95.4%) samples, and Type 3 in 60 (92.3%) samples. The sarcocysts were collected using laser microdissection, the DNA extracted from them was PCR-amplified, and their 18S ribosomal RNA and cytochrome c oxidase subunit 1 genes were sequenced. Phylogenetic analysis showed that, for both genes, each morphological sarcocyst type (Types 1, 2, and 3) aligned most closely with S. silva/S. truncata, S. tarandi/S. elongata, and S. pilosa, respectively. Based on the sequence identities between taxa and the molecular information for sarcocysts in C. nippon centralis, the sarcocyst types were presumed to be S. truncata-like (Type 1), S. tarandi-like (Type 2), and S. pilosa (Type 3). The phylogenetic analyses based on the present comprehensive molecular characterization of three Sarcocystis spp. from C. nippon yesoensis in Hokkaido suggest that canids (e.g., wild foxes) may be the definitive hosts for S. pilosa, and felids (or unknown species) the definitive hosts for the other two species.

Experimental studies on Echinococcus multilocularis in Japan, focusing on biohazardous stages of the parasite.

Alveolar echinococcosis (AE), caused by the active growth of larval Echinococcus multilocularis mostly in the liver, is usually fatal zoonotic disease if not adequately treated. Humans become infected via oral ingestion of the parasite eggs, which are thus biohazardous to humans and should be handled under restricted conditions. In this review, we present findings in experimental studies mainly performed at a safety facility in Japan, examining the biohazadous stages of the parasite (Hokkaido isolate) including its egg and adult worm stages. This article deals mainly with the parasite development in various experimental and wild animals, environmental factors affecting viability of the parasite eggs, and molecular biological studies on adult worms. The findings shown herein have provided a basis to better understand basic biology and natural transmission of E. multilocularis in Hokkaido, a highly endemic area of AE in northern Japan, and also to establish effective preventive measures against the disease.